Human Small C-terminal Domain (Scp) phosphatases are recently identified enzymes that regulate the phosphorylation states of the C-terminal domain of RNA polymerase II. More importantly, the Scp phosphatases have also been shown to inhibit the inappropriate differentiation of neuronal stem cells. In human neuronal stem cells and non-neuronal tissues, Scps strongly associate with the REST/NRSF neuronal silencing complex that functions to inhibit transcription of neuronal genes. Therefore, chemical compounds inhibiting the phosphatase activity of Scps can be powerful tools to direct neurogenesis and promote the regeneration of neurons. A long-term goal is development of a therapeutic agent that, by inhibiting Scp phosphatases, would facilitate neuronal differentiation to repair nervous system damage. Such compounds would eventually benefit patients with neuron degenerative diseases and neuronal injuries. In this proposal, we would identify potent and specific chemical agents to inactivate Scp phosphatases in malachite green phosphatase assay using the natural substrates of the enzyme. The hits would be confirmed using another two assays, fluorescence anisotropy and cell-based transcription assays. The identified hits will be used in x-ray structural analysis which will provide essential information for compound optimization and help us to improve the selectivity and affinity of inhibitors. PUBLIC HEALTH RELEVANCE: The aim of this project is to develop an inhibitor of Human Scps. Inhibition of Scps leads to neuronal differentiation from stem cells and expression of neuronal genes. Such an inhibitor will be an important reagent for studying nervous system development, as it may be used to differentiate endogenous adult neuronal stem cells to replace and repair neuronal damage, especially in the hippocampus and spinal cord. The clinical goal is to develop a novel therapy for degenerative nervous system diseases.